RNA interference was discovered in nematodes in the late 1990s, which blocks the activity of genes by using small interfering (siRNA) or small hairpin RNA (shRNA) molecules. RNAi has emerged as a powerful tool used in thousands of laboratories worldwide to understand gene function. By knocking down a genes function, RNAi can tell us about the role of any gene in maintaining health or causing disease, an invaluable step in identifying potential drug targets. In tests called genome-wide RNAi screens, scientists use automation to introduce siRNA/shRNAs into human cells to knock down the activity of each gene, one at a time. This process can produce a complete list of all genes involved in a particular biological function or disease process. Scientists also can use these techniques to understand what roles genes play in drug effectiveness. The CRISPR/Cas9 system is a form of acquired immune system found in prokaryotes, which helps bacteria resist foreign genetic elements such as bacterial phages. CRISPR RNAs (crRNAs) express in the host genomes bind to the Cas9 nuclease and guide the complex to its target DNA sequence adjacent to PAM (photospacer adjacent motifs). The CRISPR complex then cut both strains of foreign DNA to destroy the invader. In 2012, the bacterial CRISPR/Cas9 system was transformed into a genome-editing tool. Using the CRISPR/Cas9 technology, scientists are now able to modify any genome by either generating random mutations through the error-prone repair mechanism or supplying the DNA template of their choosing to knock in any gene of their interests or correct a mutation by non-homologous recombination. CRISPR/Cas9 system has also been developed as a robust reverse-genetic screening platform. In genetic screening, unlike RNAi knocks down gene expression, CRISPR/Cas9 generates completely loss-off-function phenotypes which can serve as a complementary tool for RNAi. RNAi and CRISPR/Cas9s potential usefulness in genetic screening has been limited by the lack of expertise to perform genome-scale screens, the lack of methodologies that can properly interpret these experiments and the absence of comprehensive RNAi data in public databases for researchers to reference. To address these problems, NCATS operates a state-of-the-art functional genomic screening facility known as the Trans-NIH RNAi Facility (TNRF), and NCATS staff assists NIH intramural investigators with all stages of project planning and execution. The initiative provides public access to functional genomic screening data generated from these experiments through the National Library of Medicines PubChem database. In addition, siRNA/CRISPR RNA sequence information is available from private-sector biotechnology partners. For instance, researchers can access Life Technologies Silencer Select siRNA library, which includes 65,000 siRNA sequences that target more than 20,000 human genes. TNRF, administered by NCATS Division of Pre-Clinical Innovation staff, offers a robotic platform with integrated, automated devices for conducting all aspects of screening assays (tests), including manipulating chemicals and cells, reading the results and imaging the cells. Offline (non-robotic) devices can perform smaller-scale work from assay optimization through medium-scale screening. Investigators have the option of using several different siRNA/CRISPR libraries and other small molecules involved. For data analysis, the facility offers powerful computational tools. In addition to enabling collaborations on specific projects, TNRF staff work on developing methods that advance the science of functional genomic screening, data analysis algorithms and gene perturbation technologies for exploring gene function. As a result, they recently implemented array-based CRISPR/Cas9 and FACS-assisted pooled CRISPR/shRNA screening platforms to their facility pipeline. They are currently developing more technologies such as CRISPRi/a and secretome screening platforms in order to accelerate scientific discoveries in all aspects of biology. Project areas include cancer (drug enhancer/resistance screens, development of 3D metastasis screens, molecular targets in cancer, and cancer-related pathways), infectious diseases (viral infection and replication such as Zika virus, HIV, Ebola virus, and Hepatitis C virus), fundamental cell biology (DNA replication and reprogramming/differentiation), and other disease-related phenotypes (Parkinsons disease, diabetes, and fragile X syndrome).